The ultimate objective of the proposed research is to elucidate the mechanism and regulation of the facilitated transport of hexoses in cultured animal cells and to analyze the relationship between hexose transport and phosphorylation operating in tandem in the overall uptake process. We have developed rapid kinetic techniques, which allow uptake measurements in time intervals as short as 1.5 sec., and steady state equations which describe the relationship between substrate transport and subsequent phosphorylation. The methods allow the accurate determination of the kinetics of substrate transport and phosphorylation. With these methods we propose to further characterize the hexose transport system of untransformed and transformed cultured animal cells with respect to substrate specificity, effects of pH, temperature, insulin, sulfhydryl reactive agents, and oxidizing and reducing agents. We will analyze whether its kinetic properties conform to the simple carrier model of Eilam and Stein. We will determine the kinetic constants of zero-trans, infinite-trans, equilibrium exchange and infinite-cis transport. These constants allow the estimation of the true substrate:carrier association constant and of various resistance factors related to the "movement" of the carrier within the membrane. We also intend to extend our kinetic approach to assess the effects of inhibitors on hexose carrier movement. In other studies we will determine the kinetics of hexose transport, phosphorylation as function of growth stage, cell cycle stage, serum and hexose starvation and reversal, density dependent inhibition of growth, hormone treatment, and oncogenic transformation. The results will indicate to what extent changes in overall hexose uptake rates reflect changes in transport or metabolism capacity. We also plan to study the relation between hexose transport rate and cellular growth rate in chemostat experiments.